Production Of Heavy Chain-Only Antibodies In ?1-C_H1 Knockout Mice

Immunoglobulins,or called antibodies,are one of the most important molecules in adaptive immunity.Although conventional immunoglobulins are generally composed of two heavy chains(H chain)and two light chains(L chain),heavy chain only antibodies(HcAbs)only consist of two identical heavy chains and lack light chains.HcAbs are thought as pathological antibodies in normal organisms,but in camelids and some fishes,such as sharks and ratfishes,functional HcAbs are found to be naturally expressed.As natural camelid HcAbs have particular structures and certain advantages in antigen binding,they have been widely used in laboratory practice,molecular diagnosis and development of antibody drugs.However,most currently available HcAbs are derived from camelids,and obviously,raising camelids and complicate procedures to immunize these animals appear to be a barrier for wider application of HcAbs.We were thus thinking the possibility to produce HcAbs using genetically modified small animals such as mice.As previous studies showed that all HcAbs lacked the CH1 domain of heavy chain constant region,in this study,we set out to test whether the deletion of CH1 encoding exon of ?1 gene could allow mice to produce functional HcAbs.We first constructed a gene targeting vector to replace the endogenous ?1-CH1 exon by neo gene through homologous recombination in mouse ES cells.After genotyping of the ES cells by PCR and Southern Blot,the 129Sv/JNeo+/Neo+ mouse strain,in which the ?1-CH1 exon was replaced by neo gene,was obtained through the ES cell microinjection.However,the mouse strain was found to not express IgG1 perhaps due to the integration of neo gene.We thus deleted the exogenous neo gene using the Cre-loxP system,and obtained the HG1 mouse strain with the deletion of ?1-CH1 exon and no neo gene integration.The genotype of these mice was further confirmed by PCR and Southern Blot.The transcription of ?1 gene in HG1 mouse strain was detected by RT-PCR,which showed that the?1 gene in HG1 mouse could be transcribed normally.But the q-PCR indicated that the transcriptional level was reduced,which is about 1/4 of the level in wild type mice.Furthermore,Western Blot indicated that the secreted IgG1 antibody existed in the serum of the HG1 mice,and the IgG1 antibody in HG1 mice was confirmed to be the HcAb,as shown by using Protein L,a protein could specifically bind light chains.We next analyzed the B cells in the spleen and bone marrow in HG1 mice by FACS,which showed that the percentage of pro-B cell subsets in HG1 mice was significantly increased(p<0.05),the percentage of pro-B cells in HG1 mice was 32.91 � 3.54%compared with 22.28 � 1.76%in wild type mice.The percentage of pre-B cells was significantly reduced(p<0.05)in HG1 mice,as it was 66.33�3.71%compared to 77.24 � 1.8%in wild type mice.The percentage of immature B cells and mature B cells in HG1 mice were similar to that in wild type mice.The percentage of plasma cells in HG1 mice was significantly increased(p<0.0001),but the percentage of IgG1 expressing plasma cells was significantly reduced(p<0.001).Analysis of the variable regions indicated that the HcAb in HG1 mice tended to use some of the variable families like IGHV1,and longer CDR3.Then the concentrations of each immunoglobulin class or subclass were detected by ELISA.Compared with wild type mice,the concentration of IgG1 was significantly decreased in HG1 mice,which was about 1/3 of that in wild type mice(p<0.01).However,the levels of other immunoglobulin subsets were increased in various degrees.Subsequently,we immunized the HG1 mice with OVA antigen,which comfirmed that the IgG1-HcAb was functional,and that the antigen-specific IgG 1-HcAb was produced after immunization.However,compared with wild type mice,the titer of antigen-specific IgG1-HcAb was lower(200,928.3 U/mL vs 3,212,742 U/mL),indicating that the HcAb in HG1 mice had a poor immune response to the antigen.We further constructed a phage library of the variable region of HcAb in HG1 mice which were immunized with antigen OVA.Twelve unique sequences of the variable region from HcAb were acquired by bio-panning.Based on the sequences of amino acid of CDR3,the key region for antigen binding,only 5 of 12 sequences were chosen to be expressed in E.coli.Five nanobodies were acquired,including 8#,10#,18#,35#and 42#.The abilities of the 5 nanobodies to bind OVA were detected by ELISA,only 10#and 35#nanobody showed a definite ability to recognize the antigen,and the binding ability of 10#nanobody was the best.We then used biolayer interferometry to determine the dissociation curve of antigen OVA with 10#nanobody,which indicated that 10#nanobody could bind OVA well.However,compared with the commercial monoclonal antibodies,the specificity and affinity of 10#nanobody for OVA binding appeared to be weaker.In conclusion,by deleting the ?1-CH1 exon in this study,we showed that the genetically modified mice could produce HcAbs,albeit with weaker specificity and affinity.The study in this thesis confirms the possibility to produce HcAbs using genetically modified mice,and provides a potential alternative for derivation and wider application of HcAbs.